Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates
Kendrick B. Turner, Jennifer Naciri, Jinny L. Liu, George P. Anderson, Ellen R. Goldman, Dan Zabetakis
Next-Generation Sequencing and bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library. In this work, we constructed a cDNA library of single domain antibodies from a llama immunized with staphylococcal enterotoxin B. The resulting library was sequenced, resulting in approximately 8.5 million sequences with 5.4 million representing intact, useful sequences. The sequenced library was interrogated using sequences of known SEB-binding single domain antibodies from the library obtained through phage display panning methods in a previous study. New antibodies were identified, produced, and characterized, and were shown to have affinities and melting temperatures comparable to those obtained by traditional panning methods. This demonstrates the utility of using NGS as a complementary tool to phage-displayed biopanning as a means for rapidly obtaining additional antibodies from an immune library. It also shows that phage display, using a library of high diversity, is able to select high quality antibodies even when they are low in frequency.
Circular dichroism, Secondary structure, Thermal stability, Protein folding, Protein denaturation, Thermodynamics, Antibodies, Biochemistry